This application claims the benefit of U.S. Provisional Application No. 60/360,689 filed Feb. 28, 2002.
This invention was made with government support under National Institutes of Health Grants CA88480 and CA67166 and under National Institutes of Health Cancer Biology Training Grant CA09302. The government has certain rights in the invention.
Molecular oxygen (O2) is vital to nearly all forms of lives on earth perhaps via its role in energy homeostasis, embryogenesis and differentiation. In response to hypoxia or low O2 tensions, mammals increase the expression of a wide variety of genes including erythropoietin, vascular endothelial growth factor (VEGF) and glycolytic enzymes to stimulate erythropoiesis, angiogenesis, and glycolysis (Bunn and Poyton, 1996). Most of these hypoxia-regulated genes are transcriptionally induced by the hypoxia-inducible factor-1 (HIF-1), a member of the basic helix-loop-helix Per, AhR and Sim (bHLH-PAS) family (Semenza and Wang, 1992; Wang et al., 1995a). Under normoxia, HIF-1α protein becomes hydroxylated at proline-564 in its O2-dependent degradation domain (Ivan et al., 2001; Jaakkola et al., 2001), and is targeted by the von Hippel-Lindau (VHL) protein for proteosome-mediated degradation (Maxwell et al., 1999; Ohh et al., 2000). Under hypoxia, HIF-1α becomes stabilized, translocates to the nucleus, and dimerizes with the O2-independent HIF-1β to initiate gene expression (Jewell et al., 2001; Kallio et al., 1997). The importance of cellular responses to hypoxia in development and differentiation is demonstrated in mouse models in which homozygous deletion of either HIF-1α or HIF-1β is embryonically lethal. The HIF-1α-/- embryos succumb between 9 and 10 days post coitus (d.p.c) to loss of mesenchymal cells and impaired vascular development (Iyer et al., 1998; Ryan et al., 1998). The HIF-1-/- embryos die by 10.5 d.p.c due to vascular deficiencies in the yolk sac and/or placenta (Kozak et al., 1997; Maltepe et al., 1997). Interestingly, mice heterozygous for HIF-1α exhibit increased weight loss when subjected to chronic hypoxia (Yu et al., 1999), reinforcing the essential and complex role HIF-1α plays in cellular homeostasis in a low O2 environment.
During the first trimester, a human embryo is located in a low O2 environment (3% O2) (Rodesch et al., 1992). In rat embryos, O2 tensions are low before 9.5 d.p.c (Mitchell and Yochim, 1968). The establishment of uteroplacental circulation relies on cytotrophoblast invasion into the uterine spiral arterioles. Studies indicate that cytotrophoblasts proliferate with a poorly differentiated phenotype at low O2 tensions and differentiate into a highly invasive phenotype at high O2 tensions (Caniggia et al., 2000; Genbacev et al., 1997). High O2 tensions also favor terminal differentiation of megakaryocytes into platelets (Mostafa et al., 2000). In contrast, differentiation of other cell types seems to prevail at lower O2 tensions. At 3% O2, rat mesencephalic precursor cells exhibit higher growth rates and higher levels of differentiation into a dopaminergic phenotype than at 20% O2 (Studer et al., 2000). Low O2 tensions have been found to promote osteochondrogenesis. Mesenchymal stem cells from rat bone marrow display enhanced colony-forming capability and increased proliferation at 5% O2 as compared to 20% O2, and they produce more osteocytes when implanted in vivo (Lennon et al., 2001). These observations suggest that the effect of O2 on cell differentiation is extensive and cell-type specific.
Peripheral evidence in the literature supports a role of HIF-1 in adipogenesis. Using a subtraction cloning approach, Imagawa et al. (1999) found that HIF-1α mRNA is transiently induced in 3T3-L1 (L1) preadipocytes upon treatment with the adipogenic hormone cocktail containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine (DM). However, the consequence of such transient HIF-1α expression was never investigated. Interestingly, adipogenesis can be inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which requires the aryl-hydrocarbon receptor (AhR), also a member of the bHLH-PAS family (Alexander et al., 1998; Phillips et al., 1995). Since AhR activates gene transcription by dimerization with HIF-1β (Probst et al., 1993), it is a reasonable hypothesis that inhibition of adipogenesis may be a function shared by some members of the bHLH-PAS family such as HIF-1α/β and AhR/HIF-1β.
Pathophysiological evidence exists that suggests a correlation between hypoxia and adipogenesis. For example, children with cyanotic heart disease have less body fat due to apparent adipocyte hypocellularity (Baum and Stern, 1977). High altitude training is well known to cause weight loss that is attributed in large part to body fat reduction (Armellini et al., 1997; Westerterp et al., 1994a). Strenuous physical training, on the other hand, is also attributed to significant loss of body fat (Van Etten et al., 1994; Westerterp et al., 1994b). Besides other physiological changes, hypoxia occurs in exercising skeletal muscles, as characterized by an increase in the expression of HIF-1 and VEGF (Gustafsson and Kraus, 2001; Gustafsson et al., 1999). Under hypoxia, fatty acid oxidation is impeded and glycolysis is augmented to maintain energy homeostasis. If the stored fat is not used under hypoxia, there is less need to increase or renew adipose tissue via adipogenesis. Experimentally, rats exposed to hypoxia experience significant fat loss (Mortola and Naso, 1997; Tanaka et al., 1997). Thus, reduction of adipose tissues can be caused by tissue hypoxia.
Adipocyte differentiation results from sequential induction of transcription factors C/EBPβ, C/EBPδ, PPARγ, and C/EBPα (Rangwala and Lazar, 2000; Rosen and Spiegelman, 2000). C/EBPβ and C/EBPδ are induced immediately but transiently upon IDM treatment to mediate the expression of PPARγ and C/EBPα (Christy et al., 1991; Wu et al., 1995; Yeh et al., 1995). In contrast to C/EBPδ, C/EBPβ is able to induce spontaneous differentiation in L1 cells and enhance the adipogenic potential in NIH-3T3 fibroblasts (Wu et al., 1995; Yeh et al., 1995). Highly specific for adipose tissues, PPARγ plays a critical role in the expression of most adipocyte-specific genes (Tontonoz et al., 1995) and is able to convert non-adipogenic mesenchymal cells such as fibroblasts and myoblasts to adipocytes (Hu et al., 1995; Tontonoz et al., 1994). Although developmentally necessary for adipogenesis (Wang et al., 1995b), C/EBPα is not always expressed during adipocyte differentiation especially in cells that already express C/EBPβ. For example, C/EBPα is not involved in the expression of GLUT-4, the insulin-responsive glucose transporter, in 3T3 cells ectopically expressing C/EBPβ and C/EBPδ (Wu et al., 1998). These data suggest the PPARγ and C/EBPβ may be potential targets for adipogenic intervention.
The effects of hypoxia are manifested by HIF-1 regulated genes. We and others have identified a hypoxia-induced gene DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split (HES) family of bHLH transcription factors (Ivanova et al., 2001). The HES proteins play important roles in cell differentiation by repressing gene expression (Kageyama and Ohtsuka, 1999; Staal et al., 2001). During embryonic development, DEC1/Stra13 is expressed in neuroectoderm, and in some mesoderm and endoderm derived structures (Boudjelal et al., 1997). In P19 embryonal carcinoma cells, overexpression of DEC1/Stra13 promotes neuronal differentiation and inhibits mesodermal and endodermal differentiation (Boudjelal et al., 1997). In differentiating L1 cells, DEC1/Stra13 expression is increased approximately 2-fold within 1 hr of IDM treatment, followed by a rapid decrease within 24 hr (Inuzuka et al., 1999). At present, the role of DEC1/Stra13 during adipogenesis is ill defined.
Given the importance of O2 sensing in embryonic development, as well as energy homeostasis and cell differentiation, O2 tensions may control adipose tissue function by regulating adipogenesis. Since fatty acid metabolism requires mitochondrial respiration, hypoxia prevents the use of fatty acids and thus may obviate the need for more adipose tissue. Therefore, we have investigated whether hypoxia inhibits adipogenesis through the HIF-1 dependent induction of the DEC1/Stra13 gene expression. As mentioned above, adipocyte differentiation in vitro is determined by precisely orchestrated expression of the C/EBPs and PPARγ. We have thus determined whether the C/EBP family or PPARγ is the critical target of DEC1/Stra13, and whether overexpression of DEC1/Stra13 is sufficient to inhibit adipocyte differentiation. The regulation of adipogenesis by hypoxia opens new directions for research in understanding how the microenvironment regulates cell differentiation both under physiological settings as well as during the malignant progression of tumors.